P0438, a new calcium-regulated promoter

ABSTRACT

There is disclosed a new calcium-regulated promoter to be used for increasing production of extracellular enzymes, or heterologous polypeptide, a recombinant vector that includes the DNA sequence of the promoter operatively linked to a DNA encoding said enzyme or polypeptide and a host organism transformed with the recombinant vector that includes the promoter operatively linked to the DNA encoding said enzyme or polypeptide. The present invention further relates to Streptomyces expression systems and methods for expressing foreign DNA sequences in Streptomyces and for secreting to the surrounding medium polypeptides and proteins coded for by those foreign DNA sequences.

This is a division of application Ser. No. 07/989,363, filed Dec. 11,1992, U.S. Pat. No. 5,385,841.

The invention is in the field of biotechnology. More particularly, itrelates to a new calcium-regulated promoter to be used for increasingproduction of extracellular enzymes, or heterologous polypeptides, arecombinant vector that includes the DNA sequence of the promoteroperatively linked to a DNA encoding said enzyme or polypeptide and ahost organism transformed with the recombinant vector that includes thepromoter operatively linked to the DNA encoding said enzyme orpolypeptide. The present invention further relates to Streptomycesexpression systems and methods for expressing foreign DNA sequences inStreptomyces and for secreting to the surrounding medium polypeptidesand proteins coded for by those foreign DNA sequences.

The-Streptomyces are well known producers of a variety of extracellularenzymes including proteases, phosphatases, xylanases, cellulases,amylases, lipases and nucleases.

In addition, members of the genus Streptomyces produce a large number ofantibiotics, pigments and other secondary metabolites and have a complexpattern of differentiation resulting in the formation of spores. Inbatch cultures of Streptomyces there is usually a coincidence in theproduction of extracellular enzymes and the onset of antibioticproduction and pigment biosynthesis and sporulation. All of theseprocesses are repressed by nutritional conditions favoring high growthrates and are derepressed by starvation of P, C or N sources. It isunlikely that enzyme secretion, formation of secondary metabolites anddifferentiation are completely independent but respond to similartriggering mechanisms.

Several genes of Streptomyces encoding extracellular enzymes have keencloned. These include agarase from Streptomyces coelicolor,endoglycosidase H from Streptomyces plicatus, xylanases fromStreptomyces lividans, alpha-amylase from Streptomyces hygroscopius,cellulase from Strep. spA2, beta-gaiactosidase from Strep. lividans andbeta-lactamases from Strep. cacaoi, badius and fradiae.

However, the regulatory mechanisms which control expression of thesegenes are virtually unknown. In addition to specific regulatorymechanisms, such as induction of amylase by dextrins or meltotriose andcarbon metabolite regulation of amylase or agarase, general mechanismsof derepression of several extracellular enzymes are likely to occursince simultaneous production of several polymeric-substrate degradingenzymes has been observed in Streptomyces following a nutritionaldown-shift. Such transacting regulatory genes have been found inBacillus subtilis (J. BACTERIOL. 169:324-333, 1987), Bacillus natto (J.BACTEROL. 166:20-28, 1986), and Bacillus licheniformis.

Positive regulatory genes affecting enzyme synthesis and/or secretioncan be cloned by searching for increased secretion of extracellularenzymes in a poor secretory strain such as S. lividans.

Systems for expressing foreign DNA sequences in Streptomyces havepreviously been described in, for example, EP 148,552 and WO 88/07079.These systems use the endogenous promoters of extracellular enzymesproduced by Streptomyces.

The substitution of the endogenous promoters with foreign promoters hasbeen disclosed in WO 90/14426 which describes the cloning andcharacterization of a newly isolated gene, the saf gene, encoding a newpolypeptide, referred to as saf polypeptide, which modulates directly,or indirectly, expression of the genes for extracellular enzymes inStreptomyces by interacting with the control region of the structuralgenes for the extracellular enzymes.

The promoter of the saf gene has been found to be more potent: thannatural promoter of extracellular enzymes. For example, the amylase geneexpresses much greater quantities of amylase when the saf promoter issubstituted with the natural amylase promoter. Thus, the saf promotercan be used to enhance the expression of any endogenous polypeptide orprotein in place of that protein's natural promoter.

For the cloning and characterization of the saf gene, plasmid pIJ702(Katz et el., J. GEN. MICROBIOL. 129:2703-2714, 1983) was used as acloning vector. This plasmid contains the gene of tyrosinase, the enzymeresponsible for the formation of melamin from tryosine in severalspecies of Streptomyces. The mel locus in pIJ702 has been sequenced andtwo open reading frames (ORFs) have been identified: the first is theORF corresponding to the mel gene that codes for the polypeptide chainof tyrosinase, the second ORF, located upstream of the mel gene, wasnamed ORF438 (Bernan et at., GENE 37:101-110, 1985) and its role is yetunclear.

During the study carried out to locate precisely the saf gene, fragmentSstI-KpnI (432 nucleotides--saf gene without promoter) was inserted intothe BglII site of pIJ702. A surprising aspect of the study was the lackof expression of this fragment when inserted with the right orientationand its expression when inserted with the opposite direction (clockwisedirection) to ORF438. This finding implied the presence of a fragmentwith promoter activity, before the gene mel and at the BglII cloningsite, located within the ORF438, with opposite orientation.

A principal object of this invention is to provide a new promoter whichhas been identified in a DNA fragment contained within the ORF438 ofpIJ702 plasmid. The promoter activity is positively regulated by calciumions and expression of the genes for extracellular enzymes inStreptomyces, operatively linked to said promoter, is greatly increasedin the presence of CaCl₂ solutions.

It is another aspect of the present invention to provide cloningvehicles (vectors) which include said promoter operatively linked to anendogenous or foreign DNA sequence encoding a polypeptide or protein, aswell as host organisms or cells transformed with such cloning vehicles,thereby resulting in expression and secretion of the polypeptide encodedby said DNA sequence.

Another aspect of the present invention is the integration of such acloning vehicle carrying a foreign DNA sequence into the chromosomal DNAof Streptomyces.

It is yet another aspect of the present invention a process for thepreparation of an extracellular enzyme, a desired polypeptide or proteinby culturing a transformed host organism according to the presentinvention and recovering the product from the culture broth.

The new promoter contained in the ORF438 shows promoter activity inopposite orientation to that of the ORF438 and is hereinafter referredto as Po438 (Promoter opposite to the ORF438). The knowledge of itsclockwise promoter activity in pIJ702 is very important so as to avoiderroneous interpretations in gene expression studies of DNA fragmentscloned in this vector.

The Po438 promoter is the first known calcium-regulated promoter thathas been characterized in Streptomyces and can be used to enhanceexpression in Streptomyces of selected heterologous proteins byinserting the DNA fragment containing the Po438 promoter into a suitablecleavage site upstream of the gene coding for the desired protein in asuitable cloning vehicle, transforming a Streptomyces host with suchcloning vehicles and culturing the transformed bacteria in the presenceof a Ca²⁺ solution to excrete the selected protein or portion thereof.

The promoter activity of Po438 has been detected using two differentpromoter-probe vectors. The DNA fragment containing the Po438 promoterwas subcloned in two different plasmids carrying the promoterlessaminoglycoside phosphotransferase gene (neo) and the promoterlesscatechol dioxygenase gene (XylE) respectively (FIG. 1). Both genesexpressed great quantities of each enzyme in the presence of increasingconcentrations of calcium ions, thus indicating the calcium-regulatedpromoter activity of Po438.

The transcription start point in Po438 was determined through a S1nuclease mapping experiment, which situated the start point around the Cat 24 nt from the SstI site (FIG. 4).

Referring to the accompanying drawings.

FIG. 1 Construction of plasmids pULAD50 and pULAD51 carrying the 242 bpSstI-BglII fragment of pIJ702 (containing Po438) cloned in thepromoter-probe vectors pIJ487 (Ward et al., 1986) and pIJ4083.

FIG. 2 Promoter activity of the 242 bp SstI-B1II fragment pIJ702. Akanamycin gradient (o-180 ug/ml) was established on solid MM medium(Hopwood, 1967) and was streaked with S. lividans carrying the plasmidpULAD50 (A) or pIJ487 (B).

FIG. 3 Schematic representation of a region of pULAD50 carrying the 242bp SstI-BglII fragment of piJ702 and strategy followed for S1 mapping inthe diagram the bar marked with asterisk represents the labeledfragment, and the bar with S1 represents the protected fragmentobtained. Lanes A-C-G-T show the four sequence reactions for the M13mp18single strand DNA (right) and for the 242 bp SstI-BglII fragment frompIJ702 (left).

1 Protected fragment in the hybridization between the 270 ntHindIII-SstI fragment of pULAD50 and mRNA from S. lividans [pULAD50].

2 270 nt HindIII-SstI probe.

3 242 nt BglII-SstI probe.

4 Protected fragment in the hybridization between the 242 nt BglII-SstIfragment of pIJ702 and mRNA from S. lividans [pIJ702].

The-arrowheads indicate the hybridization bands corresponding to theprotected fragments.

FIG. 4 Nucleotide sequence (SEQ ID NO.1) of the DNA region in the mel.cluster that contains the ORF438 indicating its amino acid sequence. TheBglII and SStI restriction sites which flank the DNA fragment cloned inpIJ487 to construct pULAD50 are indicated. The two first amino acids ofthe tyrosinase are also shown.

→Initiation of the mel transcript according to Geistlich et al. (1989)and Leu et al. (1989).

∞→Transcription initiation from Po438 according to S1 nuclease mappingexperiments (FIG. 3) rbs, ribosome-binding site.

FIG. 5 Phosphate repression of expression from the Po438 promoter. Aneomycin gradient (0-180 ug/ml) was made on solid MM+TES buffer 825 mM,pH 7.2) (upper plate), and on the same medium supplemented with 20 MMsodium phosphate buffer, pH 7.2 (lower plate). Both plates were streakedwith a similar amount of S. lividans [pULAD50] spores.

FIG. 6 Calcium induction in Streptomyces of the promoterless XylE geneof Pseudomonas putida when placed downstream of the Po438 promoter. S.lividans [pULAD51] was grown in calcium-free R2YE liquid medium () andR2YE medium supplemented with additional CaCl₂ up to 60 mM (66 ).Catechol dioxygenase activity (Cat O₂ ase) of S. lividans transformedwith pIJ4083 (∘) was not altered when grown in R2YE medium with orwithout CaCl₂.

The work described herein was performed employing the followingmaterials and methods.

Bacterial strains and plasmids. The Streptomyces strains and plasmidsused in this study are listed in Table 1.

Media and culture conditions. Transformation of protoplasts Streptomycesstrains were grown in R2YE, minimal medium (MM) or YEMEN supplementedwith 34% sucrose and 5 mM MgCl₂ (Hopwood et al., Genectic manipulationpf Streptomyces. A LABORATORY MANUAL. The John Innes Foundation,Norwich, U.K., 1985). Liquid cultures of Streptomyces were grown intriple baffled flasks at 28° C. in a rotary shaker with an agitation of220 rpm.

Preparation and transformation of S. lividans protoplasts were asdescribed (Thompson et. al., J. BACTERIOL. 151:668-677, 1982).

DNA isolation, manipulation and DNA sequencing. Plasmid DNA was isolatedfollowing the Kieser method (Kieser et el., PLASMID 12:19-36, 1984).Digestions and ligations were monitored by agarose gel electrophoresis.The conditions for digestion with a restriction endonuclease andligation reactions were those recommended by the manufacturers.Subcloning of DNA fragments was carried out by digesting 1-2, ug ofplasmid DNA with adequate restriction enzyme(s) and the reactionproducts were separated by gel electrophoresis in low melting pointagarose (LMPA).

The nucleotide sequence was determined by the chain termination methodof Sanger (Sanger et al., PROC. NATL. ACAD. SCI. USA 74:5463-5467, 1977)using M13 clones (Messing et al., NUCL. ACIDS RES. 9:309-321, 1981).

RNA isolation and S1 nuclease mapping. RNA was isolated according toKirby (Kirby et al. BIOCHEM. J. 104:258-262, 1967) from 50 hour culturesin MM medium. For S1 mapping the DNA probes were end labelled (Maxam andGilbert, METHODS ENZYMOL. 65:499-560, 1980). RNA (40 ug) was mixed with10⁵ c.p.m. of [³² p]-end labelled DNA fragment and denatured at 85° C.for 15 mins. (Favalora et el., METHODS ENZYMOL. 65:718-749, 1980).Hybridization was carried out at 60° C. for 3 h, treated with 60 unitsof S1 nuclease and the S1 digestion product was loaded onto a 7% (w/v)polyacrylamide gel containing 7M urea, and run in parallel with the M13mp18 phage and the 242 bp BglII-SstI fragment of pIJ702 sequenced by theSanger method.

Detection of catechol dioxygenase activity. For plate assays,Streptomyces transformant colonies were grown at 28° C. for 3 days andthe plates were sprayed with an aqueous solution of 0.5M catechol. Forliquid assays, Streptomyces strains were grown at 28° C. 50 ml of R2YEliquid medium without CaCl₂ or supplemented with CaCl₂ (60 mM). Atdifferent times samples were taken and catechol dioxygenase activitieswere determined as described (Ingram et al., J. BACTERIOL.171:6617-6624, 1989). Protein concentrations were determined by theBradford method by using bovine serum albumine as the standard(Bradford, ANAL. BIOCHEM. 72:248-254, 1976).

The following detailed description will illustrate the invention:

Promoter Activity of Po438

The promoter activity of Po438 has been determined through theexpression of the aminoglycoside phosphotransferase gene and thecatechol dioxygenase gene contained in two different promoter-probevectors.

Expression of the aminoglycoside phosphotransferase gene. A 242 bpBglII-SstI fragment from pIJ702 plasmid was subcloned into pIJ487 (Wardet al., MOL. GEN. GENET. 203:468-478, 1986), which carried apromoterless aminoglycoside phosphotransferase gene Expression of thisgene confers Kanamicin (km) and neomycin resistance to Streptomyceslividans. Plasmid pULAD50 was thus created (FIG. 1). S. Lividanstransformed with pULAD50 was able to grow on MM containing more than 150ug/ml of Km, whereas S. lividans transformed with pIJ487 (promoter-probevector without inserted promoter) does not grow on MM with 10 ug/ml Kmas shown in FIG. 2. This result clearly indicates that the BglII-SstIfragment of pIJ70 has promoter activity in Streptomyces, withorientation from the SStI site to the BglII site.

Expression of the catechol dioxygenase gene. The 242 bpBgl BglII-SstIfragment as in the above example wa subcloned into a differentpromoter-probe vector, the plasmid pIJ4083 which carried the XylE genefrom Pseudomonas putida, coding for the enzyme catechol dioxygenase. Thehybrid plasmid was named pULAD51 (FIG. 1). Streptomyces lividanstransformed with pULAD51 was grown in R2YE medium supplemented withCaCl₂ up to 60 Mm. Quantitation of Catechol dioxygenase activityindicated that Po438 exerts promoter activity also when insertedupstream of the XylE gene (FIG. 6).

Regulation of the Expression from Po438 by Calcium Ions

The effect of different ions on the promoter activity of Po438 has beenanalyzed. Since the level of Kanamycin resistance is dependent on theamount of salt in the growth medium (Hopwood et al., GeneticManipulation of Streptomyces. A Laboratory Manual. The John InnesFoundation, Norwich, U.K. 1985), Neomycin (Neo) was used in MM for thesestudies. In all cases MM was supplemented with TES buffer (25 mM, pH7.2) in order to avoid pH changes. Mg²⁺, Fe²⁺, Zn²⁺, Cu²⁺ cations had noeffect on the strength of Po438 (resistance to 170 ug/ml Neo on MMplates), whereas several monovalent cations (Na⁺, K⁺, Li⁺, Cs⁺, 10 mM)exerted a weak reduction of the promoter activity and there was nogrowth on MM containing more than 100 ug/ml of Neo. A reduction inpromoter activity-was also observed when the MM was supplemented withsodium phosphate buffer (20 mM, pH 7.2) as shown in FIG. 5.

Calcium ions positively regulate the promoter activity of Po438. Ca²⁺greatly increased Neo resistance of S. lividans transformed with pULAD50plasmid and a strict correlation between CaCl₂ concentration and Neolevel restistance has been shown (Table 2).

Plasmid pULAD60 represents an example of a similar plasmid with the sameneo-gene linked to a different promoter, the saf promoter, as has beendescribed in WO 90/14426. The Neo resistance of S. lividans transformedwith pULAD60 was not, however, modified when growing on MM containingdifferent concentrations of CaCl₂ (0, 10, 20, 30, 40 mM), suggestingthat there is not a post-translational stimulation of the neo geneproduct, nor a calcium inactivation of Neomycin. If the effect of Ca²⁺were not specific, the stimulating effect should be observed with allthe constructions. This demonstrates that the Po438 promoter activity isspecifically regulated by calcium ions.

Calcium induction was also observed in the expression of Xyle in S.lividans transformed with plasmid pULAD51 as reported in FIG. 6.Catechol dioxygenase activity (Cat O₂ ase) greatly increased when theR2YE culture medium was supplemented with CaCl₂ up to a 60 Mmconcentration. Catechol dioxygenase activity of S. lividans transformedwith pIJ4038 (the promoter-probe vector without the Po438 promoter) was,on the contrary, not altered when grown in R2YE medium with or withoutcalcium chloride.

Determination of the Transcription Start Point in Po438

S1 nuclease mapping experiments were carried out to determine thetranscription start point and the sequence of the Po438 promoter. A 270bp HindIII-SstI fragment isolated from pULAD50 was labelled in theHindIII 5' end and hybridized with mRNA isolated from S. lividanscarrying pULAD50. The protected fragment showed in a size of around 24nt shorter than the control probe (FIG. 3) what situated thetranscription start point around the C at 24 nt from the SstI site (FIG.4), A second S1 nuclease mapping experiment was performed with theoriginal plasmid pIJ702, which also contains the Po438 DNA region. The242 bp BglII-SstI fragment of pIJ702 was used as a probe. The fragmentwas labelled in the BglII 5' end and hybridized with mRNA isolated froms. lividans transformed with pIJ702. FIG. 3 shows that the protectedfragment was still 24 nt shorter than the probe, indicating that thetranscription initiation occurs in pIJ702 in a similar nucleotide as inpULAD50.

FIG. 4 shows the promoter region of the mel gene in ORF 438, locatedabout 30 nt upstream from the start codon of the ORF 438 (Geistlich et.al., Molecular Microbiology 3: 1061-1069, 1989; Leu et al., GENE84:267-277, 1989) as well as the Po438 promoter located in oppositeorientation to that of ORF 438 and its transcription start pointaccording to the S1 nuclease mapping experiments of this work.

While the aminoglycoside phosphtransferase gene and the catecholdioxygenase gene have been specifically exemplified, it should beunderstood that for the purpose of enhancing expression through the useof the Po438 promoter, the gene for any polypeptide or protein producedby the Streptomyces species being used can be modified by removing thenative promoter and substituting the Po438 promoter. Similarly, theforeign DNA coding for a polypeptide or protein can be inserted into anysuch endogenous gene. Preferably, however, the endogenous gene selectedwill be one which expresses the protein through the cell wall and intothe culture medium, in which case, it is important to retain thesecretion signal sequence. The foreign DNA is thus preferably insertedin such a way as to retain the secretion signals as much as possiblefrom the extracellular enzyme gene. While these signals arepredominantly on the leader sequence, there is evidence with respect toendogenous genes for extracellular enzymes that the terminal carboxylend is also important for secretion. Thus, it may be best to create afusion protein by inserting the foreign DNA into the endogenous DNA,rather than removing the transcriptional part of the endogenous DNA andsubstituting the foreign DNA.

It should also be understood that the foreign DNA sequence may be anynon-Streptomyces-derived DNA sequence encoding a protein or polypeptide,particularly one of eukaryotic or viral origin. Examples of sucheukaryotic and viral DNA sequences are sequences encoding human andanimal leukocyte interferons (IFN-alpha), fibrobtast interferons(IFN-beta), and immune interferons (IFN-gamma), human insulin, human andanimal growth and other hormones, such as corticotropin releasing factor(CRF), human serum albumin and various human blood factors andplasmignogen activators, both tissue and urokinase, hepatitis B viralcore and surface antigens, FMD viral antigens and other human animal andviral polypeptides and proteins.

                  TABLE 1                                                         ______________________________________                                        Strains and plasmids                                                                                       Source of                                        Designation                                                                              Relevant characteristics                                                                        reference                                        ______________________________________                                        S. lividans JI1326                                                                       wild type         JI                                               S. antibiaticus                                                               ATCC 11891 wild type, melanin producer                                                                     ATCC                                             pIJ702     thiostrepton resistance and                                                                     Katz et al. 1983                                            melanin.sup.+                                                      pULAD60    pIJ702 carrying the saf                                                                         WO 90/14426                                                 promoter                                                           pIJ487     promoter-probe vector for                                                                       Ward et. al. 1986                                           Strectomyces carrying the                                                     neo gene as reporter                                               pULAD50    pIJ487 carrying the 242 bp                                                                      This work                                                   SstI-BqlII fragment from                                                      pIJ702                                                             pIJ4083    promoter-probe vector for                                                     Streptomyces carrying the                                                     XylE gene as reporter                                              pULAD51    pIJ4083 carrying the 242 bp                                                                     This work                                                   SstI-BqlII fragment from                                                      pIJ702                                                             ______________________________________                                         JI, Collection of microorganisms of the John Innes Institute, Colney Lane     Norwich; NR4UH, UK; ATCC, American Type Culture Collection               

                  TABLE 2                                                         ______________________________________                                        Level of neomycin resistance of S. lividans [pULAD50]                         growing on solid MM supplemented with CaCl.sub.2                              CaCl.sub.2 concentration (mM)                                                                  Neo resistance (μg/ml)                                    ______________________________________                                         0               180                                                          10               300                                                          20               650                                                          30               1100                                                         40               1900                                                         ______________________________________                                    

References Quoted in the Figures and Table 1

GEISTLICH M., Irniger S. and Hutter R.: Localization and functionalanalysis of the regulated promoter from the Streptomyces glaucescens meloperon. Molecular Microbiology. 3 (1989) 1061-1069.

HOPWOOD. D. A.: Genetic analysis and genome structure in Streptomycescoelicolor. Bacterial. Rev. 31 (1967) 373-403.

KATZ, E., Thompson, C. J. and Hopwood, D. A.: Cloning and expression ofthe tyrosinase gene from Streptomyces antibioticus in Streptomyceslividans. J. Gen. Microbiol. 129 (1983) 2703-2714.

LEU, W. M., Wu, S. Y., Lin, J. J., Lo, S. J. and Wu Lee, Y. M.: Analysisof the promoter region of the melanin locus from Streptomycesantibioticus. Gene 84 (1989) 267-277.

WARD, J. M., Janssen, G. R., Kieser, T., Bibb, M. J., Buttner, M. J. andBibb, M. J.: Construction and characterization of a series of multi-copypromoter-probe plasmid vectors for Streptomyces using the aminoglycosidephosphotransferase gene from Tn5 as indicator, Mol. Gen. Genet. 203(1986) 468-478.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 3                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 572 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 94..531                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TCGCGGCCAACCGGTCCGGGCCGATTTCTCCCCTTCTCCTCCGGTCGATAGGTATGCGGG60                GTCGTCAACCCAACGCACCCCAGGAGGTCCCGCATGCCGGAACTCACCCGTCGT114                     MetProGluLeuThrArgArg                                                         15                                                                            CGCGCGCTCGGCGCCGCAGCCGTCGTCGCCGCCGGTGTCCCGCTGGTC162                           ArgAlaLeuGlyAlaAlaAlaValValAlaAlaGlyValProLeuVal                              101520                                                                        GCCCTTCCCGCCGCCCGCGCGGACGATCGGGGGCACCACACCCCCGAG210                           AlaLeuProAlaAlaArgAlaAspAspArgGlyHisHisThrProGlu                              253035                                                                        GTCCCCGGGAACCCGGCCGCGTCCGGCGCCCCCGCCGCCTTCGACGAG258                           ValProGlyAsnProAlaAlaSerGlyAlaProAlaAlaPheAspGlu                              40455055                                                                      ATCTACAAGGGCCGCCGGATACAGGGCCGGACGGTCACCGACGGCGGG306                           IleTyrLysGlyArgArgIleGlnGlyArgThrValThrAspGlyGly                              606570                                                                        GGCCACCACGGCGGCGGTCACGGCGGTGACGGTCACGGCGGCGGCCAT354                           GlyHisHisGlyGlyGlyHisGlyGlyAspGlyHisGlyGlyGlyHis                              758085                                                                        CACGGCGGCGGTTACGCCGTGTTCGTGGACGGCGTCGAACTGCATGTG402                           HisGlyGlyGlyTyrAlaValPheValAspGlyValGluLeuHisVal                              9095100                                                                       ATGCGCAACGCCGACGGCTCGTGGATCAGCGTCGTCAGCCACTACGAG450                           MetArgAsnAlaAspGlySerTrpIleSerValValSerHisTyrGlu                              105110115                                                                     CCGGTGGACACCCCGCGCGCCGCGGCCCGCGCTGCGGTCGACGAGCTC498                           ProValAspThrProArgAlaAlaAlaArgAlaAlaValAspGluLeu                              120125130135                                                                  CAGGGCGCCCGGCTCCTCCCCTTCCCCTCCAACTGACCTTCTCCCCCGCACTT551                      GlnGlyAlaArgLeuLeuProPheProSerAsn                                             140145                                                                        TTGGAGCACCCGCACATGACC572                                                      (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 146 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetProGluLeuThrArgArgArgAlaLeuGlyAlaAlaAlaValVal                              151015                                                                        AlaAlaGlyValProLeuValAlaLeuProAlaAlaArgAlaAspAsp                              202530                                                                        ArgGlyHisHisThrProGluValProGlyAsnProAlaAlaSerGly                              354045                                                                        AlaProAlaAlaPheAspGluIleTyrLysGlyArgArgIleGlnGly                              505560                                                                        ArgThrValThrAspGlyGlyGlyHisHisGlyGlyGlyHisGlyGly                              65707580                                                                      AspGlyHisGlyGlyGlyHisHisGlyGlyGlyTyrAlaValPheVal                              859095                                                                        AspGlyValGluLeuHisValMetArgAsnAlaAspGlySerTrpIle                              100105110                                                                     SerValValSerHisTyrGluProValAspThrProArgAlaAlaAla                              115120125                                                                     ArgAlaAlaValAspGluLeuGlnGlyAlaArgLeuLeuProPhePro                              130135140                                                                     SerAsn                                                                        145                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 219 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CCGCGGCGCGCGGGGTGTCCACCGGCTCGTAGTGGCTGACGACGCTGATCCACGAGCCGT60                CGGCGTTGCGCATCACATCGAGTTCGACGCCGTCCACGAACACGGCGTAACCGCCGCCGT120               GATGGCCGCCGCCGTGAGCGTGACCGCCGTGACCGCCGCCGTGGTGGCCCCCGCCGTCGG180               TGACCGTCCGGCCCTGTATCCGGCGGCCCTTGTAGATCT219                                    __________________________________________________________________________

We claim:
 1. A method of expressing a foreign DNA sequence inStreptomyces comprising operatively linking said foreign DNA sequence toa Streptomyces expression control sequence which comprises the calciumregulated promoter contained in SEQ ID NO:1 or to a fragment thereofhaving calcium regulated promoter activity.
 2. A method in accordancewith claim 1 wherein the foreign DNA sequence and Streptomycesexpression control sequence are inserted in the chromosomal DNA of theStreptomyces.
 3. A method in accordance with claim 1 wherein saidforeign DNA sequence is operatively linked to said Streptomycesexpression control sequence through a DNA sequence encoding at least apart of the signal sequence of a protein or polypeptide secreted fromStreptomyces, said DNA sequence encoding the signal sequence beingexpressed together with said foreign DNA sequence under the control ofsaid Streptomyces expression control sequence to afford secretion of theprotein or polypeptide coded for by the foreign DNA sequence.